1. Field of the Invention
This invention relates to methods and compounds for labeling DNA. The present invention also relates to the detection and identification of chromosomes or regions of chromosomes by hybridization of a multiplicity of different chromosome-specific probes. In particular, this invention relates to in situ hybridization of these chromosome specific probes to chromosomes from disrupted cells that have been prepared so as to leave the native chromosome structure essentially intact and to preserve the physical relationships between different chromosomes, different portions of the same chromosome or between chromosomes and other cellular structures.
2. Summary of the Related Art
In situ hybridization techniques are well known in the art, and have a variety of applications for the detection of chromosomes or regions of chromosomes. These applications include but are not limited to prenatal diagnosis, genetic mapping, somatic cell hybridization and the detection of specific chromosomal genetic markers of malignant and other diseases. Hybridization is performed in situ between chromosomal target DNA sequences and probe sequences that have been modified so as to be detectable by a variety of physical or chemical means after hybridization. Preparation of target chromosomal DNA is performed so as to result in the minimum amount of disruption of chromosomal and cellular structures and to permit hybridization with the probe. Methods for preparation of chromosomal target DNA are well known in the art. (See for example Gall and Pardue, Proc. Natl. Acad. Sci. USA 64: 600 (1969), John et al., Nature (London) 223: 582 (1969), Rudkin and Stollar, Nature (London) 265: 472 (1977)).
A large variety of hybridization probes, comprised of DNA and RNA sequences, are known in the art.
Gall and Pardue, supra, disclose the use of complementary RNA (cRNA) probes labeled with tritium-containing nucleotides for the detection of specific sequences on lampbrush chromosomes.
John et al., supra, disclose the use of cRNA probes labeled with .sup.125 I to detect DNA:RNA hybrids.
Bauman et al., Exp. Cell Res. 128: 485-490 (1980) discloses detection of ribosomal 5S genes on polytene chromosomes in D. hyaei after hybridization to labeled 5S RNA.
Malcolm et al., Ann. Hum. Genet. 45: 135-142 (1981) disclose the detection of the human B-globin gene on chromosome 11 after in situ hybridization of normal human chromosomes with a labeled complementary DNA (cDNA) probe.
Manuelidis et al., J. Cell Biol. 95: 619-625 (1982) disclose detection of mouse satellite DNA sequences in mouse chromosomes after hybridization with a labeled mouse satellite-specific DNA probe.
Landegent et al., Exp. Cell Res. 153: 61-72 (1984) disclose detection of mouse chromosomes after hybridization with cloned satellite DNA sequences.
Landegent et al., Nature (London) 317: 175-177 (1985) disclose detection of the human thyroglobulin gene at a specific chromosomal location after hybridization with labeled cosmid clone DNA.
Hopman et al., Histochemistry 85: 1-4 (1986) disclose detection of human chromosomes in mouse: human somatic cell hybrids after hybridization with labeled total human DNA and detection of mouse chromosomes after hybridization with cloned mouse satellite DNA sequences.
Landegent et al., Hum Genet. 73: 354-357 (1986) disclose the detection of a specific portion of human chromosome 4 after hybridization with a cloned genomic DNA probe, which has been genetically linked to the gene for Huntington's disease.
Moyzis et al., Chromosoma (Berlin) 95: 375-386 (1987) disclose specific detection of portions of human chromosomes 9 and 16 after hybridization with cloned human satellite DNA probes.
van Dekken and Bauman, Cytogenet. Cell Genet. 48: 188-9 (1988) disclose specific detection of human chromosome 1 after hybridization with fluorescently labeled cloned probes specific for the repetitive sequences found at the centromere and telomere.
Emmerich et al., Exp. Cell Res. 181: 126-140 (1989) disclose specific detection of human chromosomes 1 and 15 after hybridization with cloned probes to tandemly repeated human DNA.
Pieters et al., Cytogenet. Cell Genet. 53: 15-19 (1990) disclose detection of human chromosomes in individual sperm cells after hybridization with labeled chromosome-specific probes.
van Dekken et al., Cytometry 11: 153-164 (1990) disclose detection by flow cytometry of chromosomes 1 and Y in interphase and metaphase nuclei of normal human blood cells after hybridization with chromosome-specific labeled DNA probes.
Cremer et al., Cytometry 11: 110-118 (1990) disclose the detection of specific chromosomal damage induced by treatment of human peripheral lymphocytes with ionizing radiation after hybridization with labeled chromosome-specific DNA probes.
Kievits et al., Cytometry 11: 105-109 (1990) disclose the detection in human lymphocyte preparations of specific chromosomes or portions of chromosomes after hybridization with labeled total genomic DNA of mouse:human somatic cell hybrids and after pretreatment of the labeled DNA with unlabeled total human genomic DNA.
Methods for labeling DNA and RNA probes are well known in the art. These methods include enzymatic incorporation of modified nucleotides, chemical synthesis of probes containing modified nucleotides and direct modification of nucleotides. The modifications include derivatization of nucleotides fort he addition of labels or haptens, and the direct addition of labels or haptens. Traditional methods involved the incorporation of radioactive isotopes such as .sup.3 H, .sup.14 C, .sup.35 S, .sup.32 P and .sup.125 I. The inherent instability and correspondingly short useful lifetime of probes incorporating radioactive label, as well as health and waste management concerns, have prompted the adoption of a number of nonradioactive compounds for labeling probe DNA and RNA.
U.S. Pat. No. 4,833,251 discloses direct and synthetic modification of DNA probes, and teaches derivatization of DNA as an N 4! (substituted amino) cytosine. This derivatization is taught by incorporation of derivatized nucleotides into DNA synthesized enzymatically or by solid-phase chemistry. Derivatization is also taught by direct modification of single stranded DNA prepared from genomic eukaryotic DNA and from DNA cloned in prokaryotes using techniques well known to the skilled.
U.S. Pat. No. 4,828,979 discloses biotin modification of nitrogen atoms at nucleotide bases, which are involved in Watson-Crick base pairing of hybridized DNA and RNA sequences, and teaches the synthesis of probes labeled with biotin at these positions.
U.S. Pat. No. 4,626,501 teaches the derivatization of DNA and RNA sequences at adenosine and cytosine residues by conjugation of these residues at nitrogen atom positions N-6 and N-4, respectively, with the compound 3-(4-bromo-3-oxobutane 1-sulfonyl)-propionate and a number of related compounds.
Dale et al., Biochemistry 14: 2447-2457 (1975) teach the derivatization of nucleotides and polynucleotides with mercurated compounds.
Bauman et al., Exp. Cell Res. 128: 485-490 (1980) teach the chemical conjugation of fluorescent compounds directly to the 3' hydroxyl moiety of RNA molecules after treatment with sodium periodate.
Langer et al., Proc. Natl. Acad. Sci. U.S.A. 78: 6633-6637 (1981) disclose the enzymatic incorporation into probe DNA and RNA of UTP and dUTP that has been modified at the carbon atom 5 (C-5) position of the nucleotide.
Renz, EMBO J. 2: 817-822 (1983) teaches the conjugation of single stranded DNA probes with biotin or .sup.125 I labeled histone H1 protein.
Shroyer and Nakane, J. Cell Biol. 97: Abstract 377a (1983) disclose the derivatization of cDNA probes by treatment with dinitrophenol.
Tchen et al., Proc. Natl. Acad. Sci. USA 81: 3466-3470 (1984) disclose addition of the hapten 2-acetylaminofluorene to single stranded and double stranded DNA and RNA probes specifically at guanosine nucleotides.
Landegent et al., Exp. Cell Res. 153: 61-72 (1984) teach the use of 2-acetylaminofluorene labeled mouse satellite DNA probes for the in situ detection of mouse chromosomes.
Draper, Nucleic Acids Res. 12: 989-1002 (1984) teaches the derivatization of polynucleotides by bitsulfite-catalyzed transamination and discloses the labeling of derivatized polynucleotides with a fluorescent compound, nitrobenzofurazan.
Forster et al., Nucleic Acids Res. 13: 745-761 (1985) disclose labeling of DNA and RNA photochemically with a photoactive derivative of biotin.
Hopman et al., Histochemistry 85: 1-4 (1986) disclose the use of mercurated human total genomic DNA probes for in situ detection of human chromosomes.
A number of methods well known in the art have been developed for the detection of nonradioactively labeled probes. These methods include direct detection of fluorescently labeled compounds, and indirect methods, which rely on the binding of a reporter molecule that is then detected either directly or indirectly. These reporter-based methods include immunological methods, in which antibodies recognize either the target:probe hybrid molecule itself or the hapten derivatized to the probe, and affinity methods, which are based on specific interactions with hapten molecules derivatized to the probe. Detection of these reporter molecules has been achieved by the attachment of a fluorescent label, conjugation to an enzyme followed by enzymatic conversion of its substrate to a detectable product, or conjugation with electron-dense atoms, such as gold, iron or silver, and detection either visually or by electron microscopy.
Manning et al., Chromosoma 53: 107-117 (1975) disclose that biotin, covalently attached to RNA via electrostatic interaction between RNA and cytochrome c, can be detected after in situ hybridization by binding of ferritin-conjugated avidin molecules and visualization by electron microscopy.
Rudkin and Stollar, Nature (London) 317: 472-473 disclose detection of in situ hybridization by the use of fluorescently labeled antibodies against RNA:DNA hybrids.
Hutchinson et al., J. Cell Biol. 95: 609-618 (1982) disclose the use of avidin conjugated with colloidal gold to detect in situ hybridization of biotinylated probes by electron microscopy.
Manuelidis et al., J. Cell Biol. 95: 619-625 (1982) disclose the use of fluorescently labeled antibodies to detect in situ hybridization with biotinylated mouse satellite DNA probes.
Landegent et al., Hum. Genet. 73: 354-357 (1986) disclose the use of horseradish peroxidase-conjugated antibodies to detect in situ hybridization of a 2-acetylaminofluorene-conjugated cosmid probe for a Huntington's disease gene.
Hopman et al., Histochemistry 84: 169-178 (1986) disclose the use of sulfhydryl haptens to mercurated DNA probes for the immunological detection of in situ hybridization.
Garson et al., Nucleic Acids Res. 15: 4761-4770 (1987) disclose the use of streptavidin conjugated with alkaline phosphatase to detect hybridization of biotinylated probes for the human N-myc and B-NGF genes.
The prior art contains examples of a variety of enzymatic and chemical methods for labeling and derivatizing nucleic acid probes. Such probes can be detected by a large number of direct and indirect detection systems. This invention relates to methods and compounds for labeling DNA with xanthine and lower alkyl substituted derivatives of xanthine and reagents comprising a large number of different DNA sequences covalently labeled with xanthine and lower alkyl substituted derivatives of xanthine that are complementary to DNA sequences of a chromosome or a portion of a chromosome. These reagents permit the in situ detection of the chromosome. The xanthine- or lower alkyl substituted xanthine derivative-labeled DNA bound to the chromosome or other target can conveniently be detected by immunological techniques.